This proposal aims at improving our understanding of the mechanisms by which facilitator membrane transport proteins function. Our main focus is on the facilitative glucose transporter Glut1, which has been the subject of many biochemical and mutagenesis studies, and is a prototype for the "major facilitator superfamily". We have recently published a 3-D model of Glut1 structure. The quality factors and Ramachandran scores for our structure are at least as good as those for membrane proteins with known coordinates (AQP1 and Kcsa), and our structure accounts for existing evidence for Glut1. We propose to test our structure by pursuing the following specific aims. Aim #1. Further refinement and development of the Glut1 modeled structure. A) We will seek to complement the conclusions drawn from our structure with information on the dynamic characteristics of it. For this, we propose to run about 1 ns molecular dynamics simulations of Glut1 in a lipid bilayer and surrounded by solvent. B) We will then reanalyze the resulting structure, seeking to delineate the transport pathways, including potential docking, binding, and selectivity sites, and to understand their dynamic behavior. Aim #2. Cysteine mutagenesis studies on the predicted Glut1 transport pathways. Of the helices that line transport pathways, some have not been investigated by mutagenesis. Therefore, for residues predicted to line the channels, we propose to perform cysteine mutagenesis followed by tests of SH reagent accessibility and hence solvent accessibility. In addition, several pathogenic missense mutated residues appear to line either one of the channels. We will perform cysteine mutations at such locations to assess solvent accessibility. Functional assays will employ the Xenopus laevis oocyte expression system, and will include determinations of transport of substrates, kinetic analysis, and also determinations of water permeability. Aim #3. Comprehensive kinetic analysis and molecular dynamics simulation of selected mutants. It is accepted that conformational changes are an integral part of Glut1 operation. Hence, we will perform molecular dynamics simulations to assess the mobility of Glut1 and possible changes in its transport pathways. Questions include whether the structural pathway(s) for substrates (glucose, dehydroascorbic acid (DHA)) and water passage can account for their migration, and where in the structure are binding and regulatory sites.